Cell proliferation and differentiation in vivo is regulated by unique spatial interactions between cells. Spatial cues in conjunction with the topologically distinct location of specific attachment molecules, and the release of specific humoral factors, such as growth and differentiation factors, function as signals to the cell to proliferate, differentiate, migrate, remain in a resting state, or initiate apoptosis. The capacity of the cell to respond to these signaling triggers is dependent on the availability of specific cell surface and intracellular receptors. The signal transduction pathways that are stimulated by these molecules depend on the organization and structure of the cell cytoskeleton whose architecture is a function of multipoint cell surface interactions with these signaling molecules, surrounding cells, and extracellular matrix.
In designing cell and tissue culture environments, it is important to consider the cellular interactions that must be incorporated into the growth environment. Cell types, spatial cues, and chemical triggers and modulators play a significant role in regulating gene expression within interacting cells (Li et al., 2002, FASEB J., 17:97-99; Botarro et al., 2002, Ann. N.Y. Acad. Sci., 961:143-153; Kunz-Schughart et al., 2003, Am. J. Physiol. Cell Physiol., 284:C209-C219; Cukierman et al., 2001, Science, 294:1708-1712). Past advances in the practice of cell and tissue culture have been directed toward providing the biochemical and physical conditions that approximate the complex in vivo microenvironment within a tissue (Cukierman et al., 2001, Science, 23:1708-1712; Li et al., 2002, FASEB J., 17:97-99; Chiu et al., 2000, Proc. Natl. Acad. Sci. USA, 97:2408-2413). These efforts have been limited by factors that include the use of cell lines that have been continuously grown on and selected for their ability to proliferate on planar culture surfaces that lack the spatial cues and chemical triggers and modulators present in tissue in vivo.
Recent work has demonstrated that the unique micro- and nano-environments resulting from spatial organization of nanofibrils in three dimensions, such as collagen and other fibrillar elements of the extracellular matrix, is essential for tissue-like patterns of cell adherence, signal transduction, and differentiated function. The attachment and growth of cells on solid planar culture surfaces elicits a different pattern of cellular organization from that observed for cells in tissues in vivo (Walpita and Hay, 2002, Nature Rev. Mol. Cell. Biol., 3:137-141; Cukierman et al., 2001, Science, 23:1708-1712; Mueller-Klieser, 1997, Am. J. Physiol., C1109-C1123). When grown on a typical planar cell culture surface, fibroblasts, for example, assume a highly spread and adhering morphology in which the actin network located within the cytoplasm is organized into arrays of thick stress fibers. In contrast, when fibroblasts are grown within collagen gels or are observed in tissues, they are spindle-like in shape with actin organized in a cortical ring (Tamariz and Grinnell, 2002, Mol. Biol. Cell, 13:3915-3929; Walpita and Hay, 2002, Nature Rev. Mol. Cell. Biol., 3:137-141; Grinnell et al., 2003, Mol. Biol. Cell, 14:384-395). Moreover, the drug sensitivity of cancer cells grown in two dimensional cell cultures versus cancer cells grown in three-dimensional cell cultures has been shown to be considerably different; an outcome that has significant bearing on the design of cancer therapies involving chemotherapeutics (Mueller-Klieser, 1997, Am. J. Physiol., 273:C1109-C1123; Padron et al., 2000, Crit. Rev. Oncol./Hematol., 36:141-157; Jacks and Weinberg, 2002, Cell, 111:923-925; Weaver et al., 2002, Cancer Cell, 2:205-216).
A significant development in cell culture and tissue culture has been the introduction of matrices composed of non-toxic and biocompatible materials designed to serve as scaffolds and three-dimensional spatial organizers for dividing cells both in vitro and in vivo (U.S. patent application Ser. No. 20020133229; U.S. patent application Ser. No. 20020042128; U.S. patent application Ser. No. 20020094514; U.S. patent application Ser. No. 20020090725). The goal of these designs is to provide a growth surface with in vivo tissue-like geometry and micro- and nano-environments for cells to proliferate and differentiate into functioning tissue or regenerate damaged structures. These structures supporting functional cells can be utilized for a variety of applications, including repairing or replacing damaged tissue in the body and promoting the growth of new tissues and organs.
The successful preparation of three-dimensional cell and tissue culture technology, however, has predominantly been a function of the expertise within individual laboratories and the availability of sophisticated instrumentation. There is a significant need for a culture medium manufactured from simple or composite materials that provides the ease of use, uniformity, quality control, and flexibility associated with the standard tissue culture plate. In addition, the culture medium material and design may allow for the construction of layered assemblies of defined composition that more accurately reflect the organization of cell layers in tissues. A media comprising multiple layers of fine fibers separated by coarse fiber supports, such as the filter media disclosed in U.S. Pat. No. 5,672,399, does not provide an environment for growth of living cells.